Aliphatic dioic acids are versatile chemical intermediates useful as raw materials for the preparation of perfumes, polymers, adhesives, and macrolid antibiotics. Long chain α,ω-dicarboxylic acids may be chemically synthesized. Synthesis is difficult and most methods result in mixtures containing shorter chain lengths, requiring additional, extensive purification steps. Several strains of yeasts are known to excrete α,ω-dicarboxylic acids as a byproduct when cultured on alkanes or fatty acids as the carbon source. In particular, yeast belonging to the Genus Candida, such as C. albicans, C. cloacae, C. guillermondii, C. intermedia, C. lipolytica, C. maltosa, C. parapsilosis and C. zeylenoides are known to produce such dicarboxylic acids (Agr. Biol. Chem. 35: 2033–2042 (1971)). Also, various strains of C. tropicalis are known to produce dicarboxylic acids ranging in chain lengths from C11 through C18 (Okino et al., B M Lawrence, B D Mookherjee and B J Willis (eds), in Flavors and Fragrances: A World Perspective, Proceedings of the 10th International Conference of Essential Oils, Flavors and Fragrances, Elsevier Science Publishers BV Amsterdam (1988)), and are the basis of several patents as reviewed by Bühler and Schindler, in Aliphatic Hydrocarhons in Biotechnology, H. J. Rehm and G. Reed (eds), Vol. 169, Verlag Chemie, Weinheim (1984).
Studies of the biochemical processes by which yeasts metabolize alkanes and fatty acids have revealed three types of oxidation reactions: α-oxidation of alkanes to alcohols; ω-oxidation of fatty acids to α,ω-dicarboxylic acids; and the degradative β-oxidation of fatty acids ultimately to CO2 and water. Various strains of the yeast C. tropicalis are known to produce dicarboxylic acids ranging in chain lengths from C11 through C18 as a byproduct when cultured on alkanes or fatty acids as the carbon source (Okino et al., B M Lawrence, B D Mookherjee and B J Willis (eds.), in Flavors and Fragrances: A World Perspective. Proceedings of the 10th International Conference of Essential Oils, Flavors and Fragrances, Elsevier Science Publishers BV Amsterdam (1988)), and are the basis of several patents as reviewed by Bühler and Schindler, in Aliphatic Hydrocarhons in Biotechnology, H. J. Rehm and G. Reed (eds), Vol. 169, Verlag Chemie, Weinheim (1984).
In C. tropicalis, the first step in the ω-oxidation pathway is catalyzed by a membrane-bound enzyme complex (ω-hydroxylase complex) including a cytochrome P450 monooxygenase and a NADPH cytochrome reductase. This hydroxylase complex is responsible for the primary oxidation of the terminal methyl group to an alcohol in alkanes and fatty acids (Gilewicz et al., Can. J. Microbiol. 25:201 (1979)). The resultant alcohol is then converted to an aldehyde by fatty alcohol oxidase (FAO) and then to the dicarboxylic acid by an aldehyde dehydrogenase.
The genes that encode the cytochrome P450 and NADPH reductase components of the complex have previously been identified as P450ALK and P450RED respectively, and have also been cloned and sequenced (see e.g., Sanglard et al., Gene 76:121–136 (1989)). P450ALK has also been designated P450ALK1. More recently, ALK genes have been designated by the symbols CYP and RED genes have been designated by the symbols CPR and NCP. See, e.g., Nelson, Pharmacogenetics 6(1):1–42 (1996), which is incorporated herein by reference. See also Ohkuma et al., DNA and Cell Biology 14:163–173 (1995), Seghezzi et al., DNA and Cell Biology, 11:767–780 (1992) and Kargel et al., Yeast 12:333–348 (1996), each incorporated herein by reference. For example, P450ALK is also designated CYP52 according to the nomenclature of Nelson, supra. A small number of fatty alcohol oxidases have been described in the scientific literature in various yeasts, examples of which are Candida tropicalis, Kemp et al., Appl. Microbiol. Biotechnol, 29:370–374 (1988), Appl. Microbiol. Biotechnol. 34:441–445 (1991), Dickinson F M, Wadforth C, Biochem. J. 282:325–331 (1992), Vanhanen et al., J. Biol. Chem. 275:4445–4452 (2000), Candida maltosa, Blasig et al., Appl. Microbiol. Biotechnol. 28:589–597 (1988), Mauersberger et al., Appl. Microbiol. Biotechnol 37:66–73 (1992), Candida cloacae, Vanhanen et al., J. Biol. Chem. 275:4445–4452 (2000), Candida (Torulopsis) hombicola, Hommel et al. FEMS Microbiol. Lett. 70:183–186 (1990), and Candida (Torulopsis) apicola, Hommel et al., Appl. Microbiol. Biotechnol. 40:729–734 (1994).
FAO proteins and the corresponding coding sequences from Candida tropicalis are described in copending application Ser. No. 10/418,819 filed Apr. 18, 2003, originally filed as Provisional Application Ser. No. 60/374,021 on Apr. 19, 2002, which applications are incorporated by reference herein as if fully set forth.
Cytochromes P450 (P450s) are terminal monooxidases of the multicomponent enzyme system described above. In some instances, a second electron carrier, cytochrome b5 (CYTb5) and its associated reductase is involved in the ω-oxidation pathway. P450s comprise a superfamily of proteins which exist widely in nature having been isolated from a variety of organisms, e.g., various mammals, fish, invertebrates, plants, mollusks, crustaceans, lower eukaryotes and bacteria (Nelson, supra). First discovered in rodent liver microsomes as a carbon-monoxide binding pigment as described, e.g., in Garfinkel, Arch. Biochem. Biophys. 77:493–509 (1958), which is incorporated herein by reference, P450s were later named based on their absorption at 450 nm in a reduced-CO coupled difference spectrum as described, e.g., in Omura et al., J. Biol. Chem. 239:2370–2378 (1964), which is incorporated herein by reference.
P450 families are assigned based upon protein sequence comparisons. Notwithstanding a certain amount of heterogeneity, a practical classification of P450s into families can be obtained based on deduced amino acid sequence similarity. P450s with amino acid sequence similarity of between about 40–80% are considered to be in the same family, with sequences of about >55% belonging to the same subfamily. Those with sequence similarity of about <40% are generally listed as members of different P450 gene families (Nelson, supra). A value of about >97% is taken to indicate allelic variants of the same gene, unless proven otherwise based on catalytic activity, sequence divergence in non-translated regions of the gene sequence, or chromosomal mapping.
Metabolic pathways can be manipulated in an attempt to increase or decrease the production of various products or by-products. One example is the manipulation of the ω-oxidation pathway to produce greater amounts of dicarboxylic acids. See, e.g., U.S. Pat. No. 6,331,420, which discloses novel genes encoding certain cytochrome P450 and NADPH reductase enzymes of the ω-hydroxylase complex in yeast Candida tropicalis, and a method of quantitating the expression of genes. It can be helpful to monitor the levels of enzymes catalyzing these reactions, especially where the metabolic pathways are manipulated in an attempt to affect the production of certain products or by-products.
Immunoassays are frequently used in evaluating protein induction, expression and degradation. Since immunoassays involve interactions between antibodies and their targets, it is important to generate antibodies with appropriate antigen binding sites. Thus, purified proteins are generally required to generate such antibodies. It may be difficult however, to fashion immunoassays for detection of and/or monitoring membrane-bound proteins such as the enzymes involved in the ω-oxidation of fatty acids and alkanes to α,ω-dicarboxylic acids for several reasons. First, not all of these enzymes are induced at all times and their presence may be fleeting. It is also difficult to obtain pure samples of these enzymes, since different enzymes involved in the ω-oxidation of fatty acids and alkanes to α,ω-dicarboxylic acids may have the same or similar activity and similar molecular weights. Conversely, notwithstanding the fact that certain enzymes have a high degree of homology, such enzymes may have very different activities. In the absence of suitable alternatives, peptide sequencing may be required to verify the identity of the purified enzymes involved in the ω-oxidation pathway. Moreover, antibodies generated in response to one particular protein involved in the ω-oxidation of fatty acids and alkanes to α,ω-dicarboxylic acids can cross react against other proteins involved in the ω-oxidation of fatty acids and alkanes to α,ω-dicarboxylic acids. Accordingly, there remains a need for antigenic peptides useful for the production of antibodies reactive against enzymes involved in the ω-oxidation of fatty acids and alkanes to α,ω-dicarboxylic acids. Methods of using the generated antibodies in order to monitor enzyme levels and reactions involved in the ω-oxidation of fatty acids and alkanes to α,ω-dicarboxylic acids are also needed.